sup70–65 impairs nitrogen signaling for gene expression and sporulation. (a) CAR1 gene expression. RNA was extracted from sup70–65 mutant cells (strain LMD651U) growing in nitrogen-rich complex medium at 23°C or after 1 h of further incubation at 30°C, resolved, blotted, and probed for CAR1 mRNA and for ACT1 mRNA as a loading control. Vector lanes, cells harboring control vector YEp352; SUP70 lanes, cells harboring multicopy SUP70 plasmid pS4B-21. (b) Sporulation. Wild-type (strain 9A6D) (open symbols) and sup70–65 mutant cells (strain 6565D) (closed symbols) were incubated at 30°C on solid nitrogen-rich YEPD medium, then replica-plated to nitrogen-rich YEPG medium (circles) or to nitrogen-free sporulation medium (squares) as positive controls. Cell samples were suspended in water, and sporulation was determined visually; at least 100 cells were evaluated per sample.